Study On The Impact Of High-fat Diet-induced Gut Microbiota Dysbiosis On Spermatogenesis And Sperm Motility

Background A typical modem Western diet contains roughly 40%of fat.Many clinical studies have confirmed that high-fat diet,which leads to obesity and dyslipidemia,can also have a negative impact on male sperm quality,such as the decline of sperm concentration,sperm motility,sperm DNA integrity and morphological abnormalities.It has been reported that high-fat diet can change gut microbial composition,breaking microbial ecological balance within gut.Gut microbiota exist in a symbiotic relationship with the host,and play a major role in physiological functions,such as immunity and energy metabolism.Many diseases such as obesity,diabetes,hepatic cirrhosis,and colon cancer that occur at sites distant to the gut have been linked to gut microbial dysbiosis.It is still unclear whether gut microbial dysbiosis influences spermatogenesis.In our study,C57BL/6 male mice as well as clinical patients'samples were used to investigate the linkage between gut microbiota dysbiosis and male spermatogenesis.Objective To demonstrate if gut microbial dysbiosis induced by the high-fat diet will lead to low sperm qualityMethods 1.Animal Research.a.Construction of gut microbiota donor models.The donors were fed normal diet(ND)or high-fat diet(HFD)in order to build up gut microbiota donor models;b.Construction of fecal microbiota transplantation(FMT)mice model.The recipients were maintained on the normal diet.Gut microbiota collected from ND and HFD mice were orally fed into different recipients by FMT,which was conducted every two days for 15 weeks;c.Evaluation of spermatogenesis and gut microbes for the donors group.After 16 weeks of dietary intervention,sperm count and motility were evaluated with computer-aided sperm analysis(CASA),the gut microbiota compositions were evaluated by the16S-rDNA Sequencing;d.Evaluation of spermatogenesis and gut microbes for the FMT mice.After 15 weeks of FMT,sperm count and motility were accessed with CASA.Serum sex hormone and lipopolysaccharide(LPS)levels were determined by ELISA Kit.The morphology of testis was observed by Hematoxylin and eosin(H&E)staining.The gene expression levels within testis were examined by RNA-sequencing and reconfirmed by Real-Time PCR(RT-PCR)assays.The gut microbiota composition and abundance were evaluated by 16S-rDNA sequencing.2.Clinical Research.a.The seminal fluids were collected from health,oligozoospermia,asthenozoospermia,and teratozoospermia humans.The sperm qualities were detected by CASA.The seminal LPS levels were determined by Tachypleus Amebocyte Lysate;b.Serum samples were collected from health,oligozoospermia,asthenozoospemia,and teratozoospermia humans.The serum LPS levels were determined by Tachypleus Amebocyte Lysate;c.The fecal samples were collected from health,oligozoospermia,asthenozoospermia,and teratozoospermia humans.16S-rDNA sequencing was carried out to detemine the composition of gut microbiota ecosystem.Results a Compared with the ND donors,the sperm motility((59.79±1.847 vs 35.76±1.688)%,P<0.001)and sperm count((19.77±1.47 vs 12.02±1.169)million/mL,P<0.001)were both decreased in the HFD donors.The gut microbial compositions were changed;b.As for the FMT recipients,sperm count and sperm motility were both decreased significantly in high-fat diet recipients(HFD-FMT)compared with those in the normal diet recipients(ND-FMT).Serum LPS levels were significantly increased in the HFD-FMT recipients compared with those in the ND-FMT recipients.H&E staining and RT-PCR showed that the spermatocytes,spermatids and round spermatids in testis of the HFD-FMT recipients were decreased.Furthermore,RNA-sequencing of testis samples showed that the expression levels of 583 genes were significantly decreased more than 2 folds,133 genes expression levels were up-regulated more than 2 folds in the HFD-FMT recipients compared with the samples of ND-FMT recipients;c.The gut microbial 16S-rDNA sequences revealed that the abundance of Bacteroides,Prevotella,Rikenella,Lactobacillus and AF12 were significant difference between two FMT groups;d.In the HFD-FMT mice,a strong infiltration of lymphocytes and macrophages in the lamina propria of small intestine were observed,with elevation of toll-like receptor 4(Tlr4),myeloid differentiation primary response 88(Myd88).LPS were increased in the HFD-FMT mice compared with the ND-FMT mice;e.Several pro-inflammatory cytokines,such as interleukin 1b(Illb),C-X-C motif chemokine 10(Cxcl10),monocyte chemotactic protein 1(Mcpl)were significantly elevated in the epididymis of HFD-FMT mice;f.Sequencing and bioinformatics analysis of the clinical faecal samples collected from some healthy donors and the patients with asthenozoospermia,oligozoospermia and teratozoospermia revealed that the combined abundance of Bacteroides-Prevotella displayed a strong negative correlation with sperm motility(P=0.029).In contrast,the blood endotoxin levels of these clinical subjects showed a strong positive correlation with the abundance of Bacteroides(P=0.0214);g.The abundance of Prevotella copri,when it is more than 15%,presented a strong negative correlation with sperm motility(P=0.011).Conclusion The dysbiosis of gut microbiota induced by HFD was one of the primary causes for the impaired sperm production and motility.Such phenotype is likely mediated by elevated blood endotoxin,epididymal inflammation and the dysregulation of testicular gene expressions.Restoration of gut microbial ecosystem may potentially help some asthenozoospermia patients.